Erythromycine

ABSTRACT

Erythromycin E is useful as an antibiotic. The compounds is prepared by the fermentation of erythromycin with Streptomyces erythreus NRRL 3887 in suitable nutrient media.

United States Patent [191 Martin et al.

[ 51 Jan. 30, 1973 ERYTHROMYCINE Inventors: Jerry Roy Martin, Waukegan;Alma W. Goldstein, Lake Bluff, both of ill.

Assignee: Abbott Laboratories, Chicago, Ill.

Filed: Jan. 18, 1971 Appl. No.: 107,428

US. Cl. ..260/2l0 E, 195/80, 424/180 Int. Cl. ..C07g 11/00 Field ofSearch ..260/2l0 E Primary ExaminerLewis Gotts AssistantExaminer.lohnnie R. Brown AttorneyRobert Niblack 57 ABSTRACTErythromycin E is useful as an antibiotic. The compounds is prepared bythe fermentation of erythromycin with Streptomyces erythreus NRRL 3887in suitable nutrient media.

2 Claims, No Drawings O Ntcam W CH3 CH3 lo Ha H O/KO CH3 CH3 0 (31H5 0Ha CH2-O OH i 0 X CH; 0011a This compound is prepared by thefermentation of erythromycin in a nutrient media comprising acarbohydrate energy source such as a monosaccharide, e.g., glucose andcorn starch; and a source of nitrogen such as soy flour, corn steepliquor, ammonium nitrate and the like, which media has been seeded witha culture of Streptomyces erythreus NRRL 3887. It is preferred that thenutrient media comprise a buffering agent to moderate the pH as thefermentation progresses. Such buffering agents which are suitableinclude dihydrogen phosphate and alkaline earth carbonates, e.g.,calcium carbonate. The foregoing description of the compound of thisinvention will now further be illustrated by a specific example settingforth the best mode of preparing the compound.

Seed cultures of Streptomyces erythreus NRRL 3887 were prepared in amedium consisting of (in grams per liter) glucose monohydrate(Cerelose), 15.0; soybean meal, 15.0; and CaCO 1.0. The cultures wereincubated at 32 C. for 72 hours on a rotary shaker. The seed was addedat a level of 3-5 percent (v/v) into 500 ml. Erlenmyer flasks containing50 ml. of a fermentation medium consisting of the following componentsingrams:

Corn starch 15.0 Soybean meal 20.0 Corn steep 50. CaCO, 1.0 Soybean oil(Edsoy) 50.0 Tap water 1000 The medium was adjusted to pH 6.8 withsodium carbonate prior to sterilization. The fermentation flashes wereincubatedat 32 C. in a rotary shaker (250 rpm) for 48 hours, then 25 mg.of the finely divided erythromycin A was added to each of 40 flasks.Incubation with shaking was continued for an additional 120 hours, thenthe flask contents were pooled and clarified as follows. To thefermentation contents was added, with stirring, an equal volume of anaqueous solution of 10 percent zinc sulfate followed by a volume of 0.5N sodium hydroxide sufficient to raise the of the solution to 8.5. Afilter aid, Dicalite, was added and the mixture was stirred for 5minutes. The mixture was filtered and the clear filtrate was collected.The filtrate was extracted twice with one-half volume of ethyl acetate.

The combined ethyl acetate extract was washed two times with water anddried over anhydrous MgSO, Concentration in vacuo gave 1.112 g. of darkviscous oil. The oil was added to the top of a column of SephadexLl-l-2O (2.2 X cm) prepared and eluted with methanol. Eluded fractionsmonitored by thin layer chromatography, showed three major componentsonly partially separated from one another. Thin layer chromatographiccomparison indicated that two of the components were known compounds:unchanged added erythromycin A and anhydroerythromycin A. Fractionsenriched in the third component were pooled and concentrated to drynessto give 448 mg. of pale yellow oil. This material was chromatographed ona column of silica gel according to the method of Oleinick and CorcoranJour. Biol. Chem. 244: 7 27(1969). The composition of each fraction wasdetermined by thin layer chromatography. Fractions containing only theunknown material were pooled and concentrated in vacuo to give 98.7 mg.of white solid. Phosphate salts were removed from the preparation bypassage through a column of Sephadex LH-20 prepared in methanol. Thesalt-free solids were concentrated in vacuo to give a colorless oil.Crystallization from ether-hexane gave 33 mg. of colorless prismssoftening at and melting at As the following experimental resultsillustrate, erythromycin E has a spectrum of activity similar incharacter to erythromycin A. In the table there is shown the activity oferythromycin E against a number of bacterial strains and certain othermicroorganisms.-

in addition to erythromycin A, as disclosed inxthe above example,erythromycin B and C can also be used as starting material in thedescribed fermentation with good results.

TABLE Minimum Inhibitor-Concen- Shigella sonnei 9290 100 Pseudomonasaeruginosa BMH No. 10 100(1000) Strep. pyogenes Roper 100 Strep.pyogenes Scott 100 M yco. gallisepticum S6 25 Myco. granularum 191680.05 Myco. hyorhinis 17981 50 Myco. pneumom'ae FH 0.01 Pan. redivivus Cidal 100 Trichomonas vagirialis ClMl 100 Crithidia fasciculata 100Haemophilus influenzae 9334 2.5

Pharmaceutically acceptable non-toxic salts include single entities andmixtures of these acid addition salts which include the hydrochloride,the hydrobromide, the hydroiodide, lactobionate, thiocyanate, both loweralkyl sulfates and the higher alkyl sulfatesysuch as stearyl sulfate,lauryl sulfate, and cetyl sulfate, alkyl and aryl sulfonates,phosphates, sulfates, maleate s fumarates, succinates, tartrates,citrates, stearates, and

others commonly used in the art.

Salts obtained through variation of the acid used to neutralize the basecompound and form the acid addition salt have special advantages in someinstances because of their increased stability, increased solubility,decreased solubility, ease of crystallization, or lack of objectionabletaste. Such advantages accrue because of the ease of administration andassimilation of the particular acid addition salt and such propertiesare subsidiary to the main physiological action of the individual cationwhich is independent of the character of the anion of the acid used inthe preparation of the salt. The process of obtaining the salt is astraight-forward neutralization reaction and can be carried out in anysuitable solvent through the addition of a chemical equivalent of thebase.

i The compound of this invention may be administered orally orparenterally in conventional dosage forms such as tablets, capsules,injectables and the like, which incorporate erythromycin E alone, andwith other pharmacodynamically active substances or with suitablecarriers according to accepted pharmaceutical practices. To obtainantibiotic responses against susceptible organisms, the desired dosagerange is from about to 200 mg/kg of body weight.

I claim:

1. The compound erythromycin E having the structural formula 2. Thepharmacologically acceptable acid addition salts of the compoundaccording to claim 1.

1. The compound erythromycin E having the structural formula